With chemical labeling, the specificity of genetic targeting is lost, and expression of cysteine-free mutants is not always possible, as cysteines often play an essential role in function and structure. The amount of background labeling can be determined by labeling a cysteine-free control mutant, and, if required, a small amount of unspecific labeling can be included in the analysis as long as it can be quantified ( Groulx, McGuire, Laprade, Schwartz, & Blunck, 2011). Also, cysteines that form disulfide bridges will not label, as long as no treatment with reducing agents has been applied previously ( Gagnon et al., 2005). In cells, only those cysteines that are accessible to the fluorophore during labeling need to be removed, i.e., any cysteine in the transmembrane region or on the cytosolic side may be ignored. For purified proteins, any additional cysteines need to be removed by mutation to serine (see e.g., Faure, Starek, McGuire, Berneche, & Blunck, 2012). However, it has to be taken care, that no other cysteines are accessible to labeling. For purified proteins very high labeling efficiencies close to 100% can be achieved ( Thompson, Cronin, Bayley, & Wallace, 2011), which is excellent for the subsequent analysis of the single subunit counting data (see below). To maximize the availability, cysteines are reduced by incubation with dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) for cell expression systems and purified proteins, respectively. The labeling efficiency depends on the availability and accessibility of the cysteine, to which the protein is linked. Instead, labeling efficiency and unspecific labeling have to be taken into account. ![]() Since organic dyes are typically close to 100% fluorescent, they do not have the same limitation as the fluorescent proteins. Maleimide forms a covalent link whereas methane-thio-sulfonate forms a disulfide bridge, and its labeling is therefore reversible by reducing agents. ![]() Thiol-reactive fluorophores are widely commercially available with different linkers such as maleimide, methane-thio-sulfonate or iodoacetamide. Thiol-reactive dyes are typically used for voltage-clamp fluorometry in order to determine dynamic conformational changes of ion channels ( Cha & Bezanilla, 1998 Mannuzzu et al., 1996), but introduce too much unspecific labeling to the cell surface to be useful for single subunit counting in a cell expression system. ![]() Purified proteins can directly be labeled using thiol-reactive dyes or similar chemistry (e.g., HA-tag). Rikard Blunck, in Methods in Enzymology, 2021 2.4 Chemical labeling
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